In vitro assay for carcinogens using phenotypic transformation of human cells

ABSTRACT

Disclosed is a method to evaluate the carcinogenicity of a compound using a transformation assay. The method includes contacting a compound to be tested for carcinogenicity with a test cell. The test cell has a defect in a protective cellular mechanism selected from the group of a defect in a DNA damage repair mechanism, a defect in cell cycle control, and a defect in the ability to prevent damage induced by oxygen free radicals. Cell growth is scored to identify the presence or absence of a transformation characteristic. The development of such a transformation characteristic indicates that the compound being tested is carcinogenic. Further embodiments include a method to identify tissue-specific carcinogens, a method to identify the biochemical mechanism of carcinogenicity of a compound, and a method to evaluate the anticarcinogenicity of a compound.

This application is a divisional of U.S. patent application Ser. No.08/696,695, filed Aug. 14, 1996, now U.S. Pat. No. 5,763,180, which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to assays for the identification ofcarcinogenic compounds, and particularly transformation assays and testcells useful therefor.

BACKGROUND OF THE INVENTION

The ability of organisms to prevent cells which have damaged oramplified DNA from replicating, to repair DNA damage, or to terminatecells which have irreparable DNA damage are important defenses againstcancer. Cells which have unrepaired mutations and are permitted todivide may form tumors.

Certain human diseases arise from an inherited inability to repair DNA,to prevent DNA damage or to prevent propagation of cells with damagedDNA. As a result, victims have a high rate of cancer. When these cellsare contacted by carcinogens, they are unable to repair the DNA damageand the cells are tumorigenically transformed. For example, xerodermapigmentosum, Cockayne's syndrome and trichothiodystrophy arise from aninability of a cell to perform nucleotide excision repair. These cellsare unable to repair damage from alkylating agents and other agentswhich induce bulky additions to DNA. Fanconi's anemia induces asensitivity to crosslinking agents such as nitrogen mustard, mitomycinC, and cisplatin. Cells from patients with ataxia-telangiectasia aresensitive to ionizing radiation and to oxidative stress because they areable to continue to divide despite unrepaired DNA damage. Cells frompatients with amyotrophic lateral sclerosis are unable to repairoxidative damage caused by active oxygen free radicals because they lacksuperoxide dismutase. Hereditary nonpolyposis colon cancer occurs as aresult of failure of excision repair and mismatch repair.

DNA damage occurs as a result of exposure to mutagens or as a longerterm result of exposure to genotoxic or nongenotoxic carcinogens.Mutagenic carcinogens are usually electrophiles or are capable ofmetabolic conversion to electrophiles which attack DNA causing basealteration and mutation. Nonmutagenic carcinogens induce cellproliferation and DNA synthesis by a variety of biochemical mechanismseventually resulting in genome alteration; but they are not initiallymutagenic. Some metal cations such as vanadate act as mitogens or alterprotein phosphorylation.

New chemicals are constantly produced either for consumer use or asby-products into the environment. These potential human carcinogens aretested in cultures of prokaryotes or lower eukaryotes, in living rodentsand in mammalian cells in tissue culture. Although these tests arereproducible, reliable, quick, relatively inexpensive and do notsacrifice higher animals, they are inadequate for testing humancarcinogens.

Cell transformation assays can detect both mutagenic and nonmutageniccarcinogens. Therefore, presumably, a chemical that induces or promotestransformation is a carcinogen. To investigate chemical carcinogenesisand mechanisms or transformation, several assays have been developedwhich rely on cell transformation. (See, e.g., DiPaolo, J. A. et al.(1969) "Quantitative Studies of in vitro Transformation by ChemicalCarcinogens," J. Natl. Cancer Inst., 42:867; Reznikoff, C. A. et al.(1973) "Establishment and Characterization of a Cloned Line of C3H MouseEmbryo Cells Sensitive to Postconfluence Inhibition of Cell Division,"Cancer Res. 33:3231; Kakunaga, T. (1973) "A Quantitative System forAssay of Malignant Transformation by Chemical Carcinogens Using a CloneDerived from BALB/c3T3," Intl. J. Cancer, 12:463). Transformed foci arethe endpoint in these assays.

These tests, however, suffer from lack of reproducibility fromlaboratory to laboratory, technical difficulties, and difficulties inscoring foci as there are several different types of foci. Due to thelow transformation frequency, large numbers of plates must be used toobtain statistically significant results for weak carcinogens. Moreover,these tests do not specifically test for human carcinogens, nor arethese prior tests based on the inability of test cells to repair DNA, toprevent DNA damage or to prevent propagation of cells with damaged DNA.

A few assays have been described which can be used to detect potentialmutagens in mammalian or specifically, human, cells (See, for example,Calos, 1988, U.S. Pat. No. 4,753,874, Schiestl, 1993, U.S. Pat. No.5,273,880, Reddel et al., 1989, U.S. Pat. No. 4,885,238, Skopek et al.,1981, U.S. Pat. No. 4,302,535; Harris et al., 1996, U.S. Pat. No.5,506,131; Crespi et al., 1995, U.S. Pat. No. 5,429,948; and States etal., 1993, U.S. Pat. No. 5,180,666). The assays, however, do not usetransformation as an endpoint, but rely on more complex endpoints fordetection of carcinogens which are only useful for detecting genotoxicor mutagenic carcinogens. Therefore, the assays described prior to thepresent invention are not designed to detect non-genotoxic carcinogensand/or tissue-specific carcinogens. These assays also suffer fromtechnical complexity and limited commercial availability. Moreover,these assays do not identify the biochemical mechanism ofcarcinogenicity of carcinogenic compounds. Therefore, there exists aneed for improved transformation assays for rapid and reliable screeningfor mutagenic, genotoxic, nongenotoxic and tissue-specific carcinogens.

SUMMARY OF THE INVENTION

The present invention relates to a method to evaluate thecarcinogenicity of a compound in an in vitro assay. Such a methodincludes the step of contacting a test cell having a defect in acellular mechanism for protecting such a cell from transformation with acompound to be tested for carcinogenicity. The method further includesscoring the growth of said test cell based on a transformationcharacteristic. A positive transformation characteristic indicates thatthe compound is carcinogenic. In one embodiment, a test cell having adefect in a protective cellular mechanism has a defect selected from thegroup consisting of a defect in a DNA damage repair mechanism, a defectin cell cycle control, and a defect in the ability to prevent damageinduced by oxygen free radicals. Transformation characteristics caninclude formation of foci, anchorage independence, loss of growth factoror serum requirements, change in cell morphology, ability to form tumorswhen injected into suitable animal hosts, and/or immortalization of thecell.

In another embodiment, a test cell to be used in an assay of the presentinvention can be a test cell having a defect in a component involved ina protective cellular mechanism (e.g., a cell having a defect in a tumorsuppressor gene). In another embodiment, a test cell to be used in anassay of the present invention is a human cell which is derived from apatient having a disease involving a defect in a protective cellularmechanism. In a preferred embodiment, such a disease includes xerodermapigmentosum, Cockayne's syndrome, trichothiodystrophy, Fanconi's anemia,ataxia-telangiectasia, hereditary nonpolyposis colon cancer,promyelocytic leukemia, lymphoid leukemia, myeloid leukemia, colorectalcarcinoma, amyotrophic lateral sclerosis, Li-Fraumeni syndrome, squamouscell carcinoma and Bloom's Syndrome. In yet another embodiment, a testcell to be used in an assay of the present invention is engineered tohave a defect in a protective cellular mechanism.

Another embodiment of the present invention is a method to identifytissue-specific carcinogens. Such method includes the steps ofcontacting a putative tissue-specific carcinogen with a first test cellof a first tissue-type and also with a second test cell of a secondtissue-type, the first test cell and the second test cell having thesame defect in a protective cellular mechanism. The method furtherincludes the step of scoring cell growth of the first and second testcell for a transformation characteristic, wherein a positivetransformation characteristic in either of the first or second testcells indicates that the compound being tested is carcinogenic.Furthermore, a difference in the magnitude of the transformationcharacteristic between the first and second test cell indicates that thecompound being tested is a tissue-specific carcinogen.

One embodiment of the present invention relates to a method fordetermining the biochemical mechanism of carcinogenicity of a compound.Such method includes the steps of contacting a putative carcinogen witha first test cell which has a defect in a first protective cellularmechanism, and also with a second test cell which has a defect in asecond protective cellular mechanism, wherein the first and secondprotective cellular mechanisms are different. The method furtherincludes the step of scoring cell growth of the first and second testcell for a transformation characteristic, wherein a positivetransformation characteristic in either of the first or second testcells indicates that the compound being tested is carcinogenic.Furthermore, a difference in the magnitude of the transformationcharacteristic between the first and second test cell indicates that thecarcinogen functions within a biochemical pathway that is associatedwith a specific defect in a protective cellular mechanism.

Yet another embodiment of the present invention is a method to evaluatethe carcinogenicity of a compound which includes the steps of contactinga compound to be evaluated with a test cell having a defect in aprotective cellular mechanism which is isolated from a patient having adisease selected from xeroderma pigmentosum, Cockayne's syndrome,trichothiodystrophy, Fanconi's anemia, ataxia-telangiectasia, hereditarynonpolyposis colon cancer, promyelocytic leukemia, lymphoid leukemia,myeloid leukemia, colorectal carcinoma, amyotrophic lateral sclerosis,Li-Fraumeni syndrome, squamous cell carcinoma and Bloom's Syndrome. Thestep of contacting is conducted in the presence of normal cells which donot have such a defect. The method further includes the step of scoringthe cells for the formation of foci, which indicates that the compoundis carcinogenic.

Another aspect of the present invention includes a method to identifyanticarcinogenic compounds. In one embodiment, this method includescontacting a test cell with a compound being tested foranticarcinogenicity. The test cell has a defect in a protective cellularmechanism selected from a defect in a DNA damage repair mechanism, adefect in cell cycle control, and a defect in the ability to preventdamage induced by oxygen free radicals. In one embodiment, the test cellis contacted with the putative anticarcinogenic compound in the presenceof a known carcinogen. The method further includes scoring cell growthof the test cell based on identification of a transformationcharacteristic. The absence of a transformation characteristic indicatesthat the compound being tested is anticarcinogenic.

In another embodiment of a method to identify anticarcinogeniccompounds, the test cell, which has the phenotype of being transformedin the absence of carcinogens, is contacted with the putativeanticarcinogenic compound. The absence or reduction of a transformationcharacteristic indicates that the compound being tested isanticarcinogenic.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to an in vitro assay forcarcinogens which uses phenotypic transformation of test cells as anendpoint. Such an assay involves contacting a compound being tested forcarcinogenicity with a test cell having a defect in a cellular mechanismwhich normally protects a cell from becoming transformed. Such a defectincludes a defect in a DNA damage repair mechanism, a defect in cellcycle control, and/or a defect in the ability to prevent damage inducedby oxygen free radicals. The method to evaluate the carcinogenicity of acompound further involves identifying whether the compound causestransformation of such test cell having normal growth to a cell havingabnormal or altered growth properties. Various aspects of transformedcells and identifying characteristics of transformation are discussed inmore detail below.

The method to evaluate the carcinogenicity of a compound has severaladvantages over previously described assays. A significant advantage ofthe present invention, because it involves a transformation assay, isthat it is capable of identifying nongenotoxic carcinogens as well asgenotoxic carcinogens. For example, the well-known Ames test onlydetects genotoxic carcinogens (e.g., mutagens). One option foridentifying non-genotoxic carcinogens is by animal testing. However,animal testing is relatively expensive and time-consuming.

The assay of the present invention also has the advantages over priorassays of being simple, sensitive, having short incubation times, andeasily scored endpoints. The methods of evaluating carcinogens disclosedherein are particularly useful for detecting human carcinogens. Moreparticularly, the methods disclosed herein are useful for identifyingthe biochemical mechanisms of the carcinogenic activity of a particularcompound, because such methods comprise test cells which carryparticular defects in protective cellular mechanisms, such as thosepresent in many disease states. Another advantage of the assay of thepresent invention is that tissue-specific carcinogens can be identified.

The method of the present invention provides a test cell having anenhanced transformation response in the presence of carcinogens comparedto normal or wild-type cells. Such a test cell has a defect in aprotective cellular mechanism. A protective cellular mechanism, as usedherein, is a cellular process capable of protecting a cell from becomingtransformed. According to the present invention, such a cellularmechanism includes a DNA damage repair mechanism, a cell cycle controlmechanism, and/or a mechanism involved in the ability to prevent damageinduced by oxygen free radicals. As such, a defect in a test cell of thepresent invention comprises a defect in at least one of the abovementioned protective cellular mechanisms. Examples of such protectivecellular mechanisms are described in detail below.

As used herein, the terms "defect" or "defective" refer to anabnormality or a deficiency within a protective cellular mechanism asdescribed herein. Such a defect causes a cell to be more susceptible totransformation when the cell is exposed to a carcinogen than a cell nothaving the defect (i.e., a normal, or wild-type cell). In oneembodiment, a defect in a test cell of the present invention can be adefective component within a protective cellular mechanism. Such acomponent can include, for example, a protein or a nucleic acid moleculeinvolved in a protective cellular process such as DNA repair,transcription, tumor suppression, cell cycle control, response toreactive oxygen species (e.g. oxygen free radicals), and apoptosis.

A defective component can be, for example, a protein in which aminoacids have been deleted, inserted, inverted, substituted and/orderivatized (e.g., by glycosylation, phosphorylation, acetylation,myristylation, prenylation, palmitoylation, amidation and/or addition ofglycerophosphatidyl inositol), such that the protein is incapable ofperforming its function within a cell, and therefore renders such cellsusceptible to transformation. A defective component can also be anucleic acid molecule in which nucleotides have been inserted, deleted,substituted, and/or inverted, such that the protein encoded by suchnucleic acid molecule is not transcribed, is not translated, or alteredsuch that the protein is unable to perform its function within a celland therefore renders such cell susceptible to transformation.

As used herein, a cell is considered to be transformed when, after ithas been subjected to a carcinogenic agent in cell culture, it hasdeveloped aberrant growth properties or characteristics. As used herein,the term "carcinogen" is a compound (e.g., a chemical, a protein, amolecule) which causes a cell to demonstrate transformationcharacteristics in a transformation assay of the present invention. Suchtransformation characteristics can include any properties associatedwith tumor or cancer cells. In particular, such characteristics caninclude formation of foci, anchorage independence, loss of growth factoror serum requirements, change in cell morphology, ability to form tumorswhen injected into suitable animal hosts, and/or immortalization of thecell. The presence of one or more of such transformation characteristicsis indicative that the compound being tested is carcinogenic. In apreferred embodiment of the present invention, such a transformationcharacteristic is the formation of foci.

One transformation characteristic is when a cell, which normally doesnot form a focus, forms a focus when grown on a culture dish. Suchcells, when not transformed, typically grow in a flat and organizedpattern until they cover the surface of a Petri plate with liquid mediumon top of them. Then, when each cell is touching its neighbor cell, cellgrowth stops by virtue of a phenomenon known as contact inhibition. Suchcells, when transformed, are not contact inhibited and will grow to highdensities in disorganized foci.

A further transformation characteristic which is indicative of a cellbeing transformed by a carcinogenic compound is anchorage independence.When anchorage independence is the transformation characteristic beingused in a particular assay, the cells used in the assay are cells which,when not transformed, are anchorage dependent. That is, when such cellsare not transformed, they grow only when attached to a solid surface.Upon becoming transformed, such cells will grow in a medium (e.g.semi-solid agar) without being attached to a solid surface.

A further transformation characteristic which is useful in assays of thepresent invention is the loss of growth factor or serum requirements.Cells used in assays of the present invention in which loss of growthfactor or serum requirements is the transformation characteristic, whennot transformed, require the presence of isolated growth factors orserum for growth. Upon transformation, such cells are able to grow in adecreased concentration or absence of the growth factors or serumrequired by the untransformed cells.

Another transformation characteristic is when a cell, upontransformation, exhibits a change in cell morphology. A change inmorphology refers to a change in the structure or shape of the cell ascompared to the cell when it is not transformed. Such a change can beobserved visually, such as by examining the cells with the aid of amicroscope.

A further transformation characteristic which is useful in an assay ofthe present invention is the ability of a cell to form tumors wheninjected into suitable animal hosts. When this ability to form tumors isthe transformation characteristic, such cells, when not transformed,will not form tumors when injected into animal hosts.

Yet another transformation characteristic is immortalization of a cell.A transformed cell which has the characteristic of being "immortalized"is capable of proliferating indefinitely in long-term culture in thepresence of appropriate nutrients and growth factors. That is, when thecell is not transformed, it has a limited life span in culture andeventually dies.

In one embodiment of the present invention, a test cell has a defect ina DNA repair mechanism. A DNA repair mechanism refers to a variety ofcellular mechanisms that effect the repair of DNA damage caused byextrinsic or intrinsic agents. For instance, DNA damage can be inducedby factors such as UV light, ionizing radiation, reactive oxygen speciesand electrophilic alkylating agents. As such, a test cell of the presentinvention can have a defect in the ability to repair DNA damage that isinduced by such factors. As used herein, DNA damage can also be referredto as a lesion (e.g. abnormal or modified bases, such as bases damagedby alkylation, oxidation, reduction or fragmentation).

According to the present invention, DNA repair is accomplished by avariety of mechanisms which include, but are not limited to, direct DNArepair, base excision repair, nucleotide excision repair, nucleotidemismatch repair, post-replication repair, cross-link repair,double-strand break repair, and transcription repair.

Direct DNA repair refers to the process by which the chemical bonds thatcomprise a lesion or connect a modifying agent (e.g. an alkyl group) tothe DNA are broken. Base excision repair refers to the process by whicha defective base is removed by a glycosylase and the resulting abasicsite is removed and replaced with a normal base. Nucleotide excisionrepair refers to the process by which a lesion is removed by dualexcision on either side of the lesion, followed by repair of theresulting gap in the DNA. Nucleotide mismatch repair refers to theprocess by which improper nucleotide base-pairings (e.g. an adeninepaired with a guanine) are corrected. Post-replication repair is aprocess by which a cell allows completion of replication withoutdirectly eliminating a DNA lesion. Such post-replication repair canoccur by translesion synthesis, template switching and/or recombination.Cross-link repair refers to the elimination of DNA interstrandcross-links by dual incisions to release the cross-link, followed eitherby strand transfer and ligation, or by filling of the gap by DNApolymerase involving a three strand intermediate. Double-strand breakrepair is a process whereby breaks occurring in both DNA strands arerepaired by a variety of complex reactions which can includerecombination events. Transcription repair can occur during all phasesof transcription, including activation, initiation, and/or elongation,utilizing a variety of enzymes and proteins to effect the repair.

Test cells of the present invention having a defect in a DNA repairmechanism can have a defect in any of the above-mentioned DNA repairprocesses, including a defect in any component which is involved in suchDNA repair process. More particularly, a test cell of the presentinvention can have a defect in a protein selected from the group ofKu80, DNA-PK_(CS), O⁶ -methylguanine-DNA methyltransferase, uracil DNAglycosylase, hydroxymethyluracil DNA glycosylase, thymine glycol DNAglycosylase, N-methylpurine DNA glycosylase, 8-hydroxyguanine DNAglycosylase, AP endonuclease, DNA Polβ, DNA Polε, DNA Polδ, poly(ADP-ribose) polymerase, XPA, p89/XPB-ERCC3, p80/XPD-ERCC2, p62(TFB1),p44/hssL1, p41/cdk7, p38/cyclinH, p34, XPC(p125), HHRA D23(p58), XPF,ERCC1(p33), XPG(p160), p70, p11, AP-1, NFκB, RNA PolI, RNA PolII, hMLH1,hMSH2, DHFR, HPRT, CSA, and CSB, and/or genes corresponding thereto.

Test cells of the present invention having a defect in a DNA repairmechanism are known in the art and include, but are not limited to, celllines selected from the group of TTD1BR, NIGMS GM739, TK6, MT1, NIGMSGM5509A, NIGMS GM2359, ATCC CRL1162, TTD8PV, XP3NE, and XP17PV. Inanother embodiment, such test cells include cells isolated from patientshaving diseases which can be characterized by a defect in a DNA damagerepair mechanism. For example, such test cells having a defect in a DNArepair mechanism include cells from patients with diseases such asCockayne's Syndrome, trichothiodystrophy, and xeroderma pigmentosum.

In another embodiment of the present invention, a test cell has a defectin cell cycle control. According to the present invention, a defect incell cycle control can result from cellular damage due to extrinsic orintrinsic agents. Such damage can be induced by factors such as UVlight, ionizing radiation, reactive oxygen species and electrophilicalkylating agents. As used herein, cell cycle control refers to theregulation of the growth, division, and death of cells. As such, adefect in cell cycle control can be manifested at any stage in the cellcycle, including at or during the G1 phase (i.e. first growth phase),the S phase (i.e. DNA synthesis), the G2 phase (i.e. second growthphase), and/or the M phase (i.e. mitosis). A defect in cell cyclecontrol imparts upon a test cell of the present invention thecharacteristic of having an enhanced transformation response in thepresence of carcinogens compared to normal or wild-type cells. Inaddition, a defect in cell cycle control can be manifested in thecellular control of apoptosis. Apoptosis, or programmed cell death, is amechanism employed by a cell for several purposes, including as aresponse to DNA damage or for inhibition of DNA synthesis. Apoptosis inresponse to such damage is an important means of preventingtransformation of a damaged cell.

As used herein, a defect in cell cycle control includes defects incomponents involved in cell cycle control, and in particular, apoptosis.Such components include tumor suppressors, which typically control thecell cycle by activating genes which are capable of inhibiting orblocking cell cycle progression and inducing apoptosis. Examples of suchcomponents which are involved in cell cycle control include p53,retinoblastoma (pRB), p21, Gadd45, human single-stranded DNA bindingprotein (HSSB), cdk-activating kinase (CAK), cdk7, cyclin H, and cyclinA.

Test cells of the present invention having a defect in cell cyclecontrol are known in the art and include, but are not limited to, celllines having a defect any of the above-mentioned components involved incell cycle control. In particular, such test cells include cellsselected from the group of Li-Fraumeni p₅₃ mut/mut fibroblasts, HL-60,RPM18402, KG-1a, RKO, CSA, BMA, NIGMS 718, NIGMS 3189, NIGMS 1526, NIGMS3382, and HeLa cells. In another embodiment, such test cells includecells isolated from patients having diseases which are characterized bya defect in cell cycle control. For example, test cells having a defectin cell cycle control include cells from patients with diseases such aspromyelocytic leukemia, lymphoid leukemia, myeloid leukemia, colorectalcarcinoma, and ataxia-telangiectasia.

In yet another embodiment, a test cell of the present invention has adefect in a cellular mechanism which confers the ability to preventdamage induced by oxygen free radicals. In particular, such a defectcomprises a defect in a protein involved in control of an oxidativestress response. An oxidative stress response is a cellular mechanismwhich protects against cellular injury due to active oxygen freeradicals. In a normal cell, oxygen free radicals and other reactiveoxygen species are produced as a result of oxidative stress and normalaerobic respiration, and are scavenged and detoxified to prevent damageto DNA and tissue. According to the present invention, a defect in theability to prevent damage induced by oxygen free radicals can resultfrom cellular damage due to extrinsic or intrinsic agents. Such damagecan be induced by factors such as UV light, ionizing radiation, reactiveoxygen species and electrophilic alkylating agents. A test cell having adefect in a component involved in such oxidative stress response is aparticularly preferred test cell of the present invention. Componentsinvolved in response to oxidative stress include superoxide dismutase,catalase, heat shock proteins, peroxidases, glutathione peroxidase, DTdiaphorase (NADH-NAD(P)H):quinone oxidoreductase, AP-1, and NF-kB.

Test cells having a defect in the ability to prevent damage induced byoxygen free radicals are known in the art and include cells having adefect in a component involved in the oxidative stress response such asthose listed above. Particularly preferred test cells having a defect inthe ability to prevent damage induced by oxygen free radicals areselected from the group of NIGMS CS1AN and NIGMS GM1856. In oneembodiment, test cells having a defect in the ability to prevent damageinduced by oxygen free radicals include cells from patients withdiseases which are characterized by such a defect. Such diseases includeCockayne's syndrome, ataxia-telangiectasia, and amylotrophic lateralsclerosis. In one embodiment, a test cell of the present invention isderived from a patient having a disease in which such disease isinfluenced or characterized by a defect in one of the protectivecellular mechanisms described herein. Such diseases include, forexample, xeroderma pigmentosum, Cockayne's syndrome,trichothiodystrophy, Fanconi's anemia, ataxia-telangiectasia, hereditarynonpolyposis colon cancer, promyelocytic leukemia, lymphoid leukemia,myeloid leukemia, colorectal carcinoma, amyotrophic lateral sclerosis,Li-Fraumeni syndrome, squamous cell carcinoma and Bloom's Syndrome.

According to the present invention, a test cell is preferably aeukaryotic cell, and more preferably, a mammalian cell. In a mostpreferred embodiment, a test cell of the present invention is a humancell. A test cell of the present invention can be a cell with anaturally occurring defect in an above-described protective cellularmechanism. For instance, a test cell with such a naturally occurringdefect can be isolated from a patient having a disease which ischaracterized by a defect in a protective cellular mechanism asdescribed above. In a further embodiment, a test cell can be engineeredto have a defect in a protective cellular mechanism (i.e., such a defectis not naturally occurring). Such a cell into which a defect isengineered, or induced, can be a modified cell or a recombinant cell. Inone embodiment, a modified cell includes a cell wherein a defect in aprotective cellular mechanism is induced by incorporation of a portionof a virus genome into a nucleic acid molecule which encodes a proteininvolved in such mechanism. It is within the scope of the presentinvention that a cell with a naturally occurring defect in a protectivecellular mechanism can additionally be modified or used as a host cellfor a recombinant molecule to induce, enhance, delete, or add furtherdefects in a protective cellular mechanism of the present invention.

As used herein, a modified cell is a cell into which a defect has beenengineered or induced such that the cell is different from the cellbefore such manipulation (i.e. different from the normal, or wild-typecell). A modified test cell, for instance, has a genome in which aportion or portions of the test cell genome involved in a protectivecellular mechanism as described herein is modified in such a way as tomake the test cell more susceptible to transformation. For example, thegenome of a cell which normally does not have a defect in a protectivecellular mechanism as described herein can be modified to induce, orcreate, such a defect. Nucleic acid molecules within a normal cell canbe modified using a variety of techniques including, but not limited to,classic mutagenesis techniques and recombinant DNA techniques, such assite-directed mutagenesis, chemical treatment of a nucleic acid moleculeto induce mutations, restriction enzyme cleavage of a nucleic acidfragment, ligation of nucleic acid fragments, PCR amplification and/ormutagenesis of selected regions of a nucleic acid sequence, synthesis ofoligonucleotide mixtures and ligation of mixture groups to "build" amixture of nucleic acid molecules and combinations thereof. In oneembodiment, a defective homologue of a nucleic acid molecule whichencodes a protein involved in a protective cellular mechanism can beproduced using a number of methods known to those skilled in the art(see, for example, Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Labs Press.) The referenceSambrook et al., ibid., is incorporated by reference herein in itsentirety. Nucleic acid molecule homologues can be selected from amixture of modified nucleic acids by screening for the function of theprotein encoded by the nucleic acid.

A recombinant cell is a type of modified cell that is preferablyproduced by transfecting a host cell with one or more recombinantmolecules, each comprising one or more isolated nucleic acid moleculesencoding proteins involved in a protective cellular mechanism of thepresent invention which have a defect as described previously herein.Such a recombinant cell can include cells in which the correspondingendogenous protein involved in the protective cellular mechanism hasbeen modified such that the endogenous protein is also defective. Inanother embodiment, a recombinant cell includes cells in whichoverexpression of an endogenous protein involved in a protectivecellular mechanism by transfection with a recombinant molecule encodingsuch protein, even if the endogenous protein itself is not modified,results in a defect in such protective cellular mechanism.

As used herein, isolated nucleic acid molecules encoding a defectiveprotein involved in a protective cellular mechanism are operativelylinked to an expression vector containing one or more transcriptioncontrol sequences. The phrase operatively linked refers to insertion ofa nucleic acid molecule into an expression vector in a manner such thatthe molecule is able to be expressed when transfected into a host cell.As used herein, an expression vector is a DNA or RNA vector that iscapable of transfecting a host cell and of effecting expression of aspecified nucleic acid molecule. Preferably, the expression vector isalso capable of replicating within the host cell. Expression vectors canbe either prokaryotic or eukaryotic, and are typically plasmids.Expression vectors of the present invention include any vectors thatfunction (i.e., direct gene expression) in recombinant cells of thepresent invention, including in mammalian cells.

The term isolated nucleic acid molecule can include an isolated naturalgene which has a defect such that it encodes a defective proteininvolved in a protective cellular mechanism described herein, such as aprotein involved in DNA repair, or a homologue thereof. Such a nucleicacid molecule can include one or more regulatory regions, full-length orpartial coding regions, or combinations thereof. The minimal size ofsuch a nucleic acid molecule is the minimal size that encodes for aprotein which is involved in a protective cellular mechanism. It is tobe noted that the term "a" or "an" entity refers to one or more of thatentity; for example, an isolated nucleic acid molecule refers to one ormore isolated nucleic acid molecules or at least one nucleic acidmolecule. As such, the terms "a" (or "an"), "one or more" and "at leastone" can be used interchangeably herein. It is also to be noted that theterms "comprising", "including", and "having" can be usedinterchangeably.

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subject to human manipulation). Forexample, an isolated nucleic acid molecule can be a gene which has beenseparated from other genes with which it naturally occurs. As such, theterm isolated does not reflect the extent to which the nucleic acidmolecule has been purified. An isolated nucleic acid molecule caninclude DNA, RNA, or derivatives of either DNA or RNA.

An isolated nucleic acid molecule can also be produced using recombinantDNA technology (e.g., polymerase chain reaction (PCR) amplification,cloning) or chemical synthesis. Isolated nucleic acid molecules includenatural nucleic acid molecules and homologues thereof, including, butnot limited to, natural allelic variants and modified nucleic acidmolecules in which nucleotides have been inserted, deleted, substituted,and/or inverted in such a manner that such modifications do notsubstantially interfere with the nucleic acid molecule's ability toencode a protein which is involved in a protective cellular mechanism.

According to the present invention, an isolated nucleic acid moleculeincludes a nucleic acid sequence that encodes at least one defectiveprotein described in the present invention. Although the phrase "nucleicacid molecule" primarily refers to the physical nucleic acid moleculeand the phrase "nucleic acid sequence" primarily refers to the sequenceof nucleotides on the nucleic acid molecule, the two phrases can be usedinterchangeably. Proteins of the present invention include, but are notlimited to, proteins having full-length naturally occurring codingregions, proteins having partial coding regions, fusion proteins, andcombinations thereof.

The use of a recombinant cell, as described herein, includes the use ofa recombinant vector, which comprises at least one isolated nucleic acidmolecule encoding a defective protein involved in a protective cellularmechanism, which is inserted into any vector capable of delivering thenucleic acid molecule into a test cell. Such a vector containsheterologous nucleic acid sequences, that is nucleic acid sequences thatare not naturally found adjacent to nucleic acid molecules encoding adefective protein, and that may be derived from a species other than thespecies from which the nucleic acid molecule(s) are derived. The vectorcan be either RNA or DNA, either prokaryotic or eukaryotic, andtypically is a plasmid or a virus and preferably is a plasmid. One typeof recombinant vector, referred to herein as a recombinant molecule anddescribed in more detail below, can be used in the expression ofdefective proteins of the present invention.

In an assay of the present invention in which the test cell beingcontacted with a putative carcinogen is a recombinant cell, therecombinant cell is cultured such that the cell is capable of expressinga defective protein involved in a protective cellular mechanism asdescribed herein, under conditions effective to produce the protein. Itshould be noted that such a recombinant cell may be repeatedly clonedand selected until such nucleic acid molecule or molecules have stablyintegrated into the host cell genome. Transfection of a nucleic acidmolecule into a cell can be accomplished by any method by which anucleic acid molecule can be inserted into the cell. Transfectiontechniques include, but are not limited to, electroporation, CaCl2precipitation, microinjection, lipofection, adsorption, and protoplastfusion.

Transfected nucleic acid molecules of the present invention can remainextrachromosomal or can integrate into one or more sites within achromosome of the transfected (i.e., recombinant) cell in such a mannerthat their ability to be expressed is retained. Preferably, once a hostcell of the present invention is transfected with a nucleic acidmolecule, the nucleic acid molecule is integrated into the host cellgenome. A significant advantage of integration is that the nucleic acidmolecule is stably maintained in the cell. The nucleic acid molecule canbe integrated into the genome of the host cell either by random ortargeted integration.

When the test cell of the present invention is a recombinant cell,suitable host cells to transfect include any cell that can betransfected with a nucleic acid molecule encoding a defective proteininvolved in a protective cellular mechanism, including any eukaryoticcell, and, preferably, the host cells are mammalian, and even morepreferably, the host cells are human. Host cells can be eitheruntransfected cells or cells that are already transfected with at leastone nucleic acid molecule. Host cells can be any cell capable ofexpressing at least one protein involved in a protective cellularmechanism. Preferred host cells include fibroblasts, epithelial cells,neurons, hepatocytes, keratinocytes, hematopoietic cells and other bonemarrow cells, kidney cells, and lung cells.

As used herein, recombinant molecules used in a recombinant test cell ofthe present invention may also (a) contain secretory signals (i.e.,signal segment nucleic acid sequences) to enable an expressed protein asdescribed herein to be secreted from the cell that produces the proteinand/or (b) contain fusion sequences which lead to the expression ofnucleic acid molecules encoding such proteins as fusion proteins.Recombinant molecules may include intervening and/or untranslatedsequences surrounding and/or within the nucleic acid sequences of suchnucleic acid molecules.

Nucleic acid molecules can be operatively linked to expression vectorscontaining regulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell andthat control the expression of such nucleic acid molecules. Inparticular, recombinant molecules include transcription controlsequences. Transcription control sequences are sequences which controlthe initiation, elongation, and termination of transcription.Particularly important transcription control sequences are those whichcontrol transcription initiation, such as promoter, enhancer, operatorand repressor sequences. Suitable transcription control sequencesinclude any transcription control sequence that can function in arecombinant test cell. A variety of such transcription control sequencesare known to those skilled in the art. In one embodiment, suchtranscription control sequences can be derived from the same source asthe isolated nucleic acid molecule. Transcription control sequences canalso include naturally occurring transcription control sequencesnaturally associated with a nucleic acid molecule prior to isolation.

It will be appreciated by one skilled in the art that use of recombinantDNA technologies can improve control of expression of transfectednucleic acid molecules by manipulating, for example, the number ofcopies of the nucleic acid molecules within a test cell, the efficiencywith which those nucleic acid molecules are transcribed, the efficiencywith which the resultant transcripts are translated, and the efficiencyof post-translational modifications. Recombinant techniques useful forcontrolling the expression of nucleic acid molecules include, but arenot limited to, integration of the nucleic acid molecules into one ormore test cell chromosomes, addition of vector stability sequences toplasmids, substitutions or modifications of transcription controlsignals (e.g., promoters, operators, enhancers), substitutions ormodifications of translational control signals (e.g., ribosome bindingsites, Shine-Dalgarno sequences), modification of nucleic acid moleculesto correspond to the codon usage of the test cell, and deletion ofsequences that destabilize transcripts. The activity of an expressedrecombinant protein may be improved by fragmenting, modifying, orderivatizing nucleic acid molecules encoding such a protein.

In accordance with the present assay, test cells with naturallyoccurring defects in a protective cellular mechanism or recombinant testcells having such defects are seeded on a culture dish in medium underconditions which promote cell growth and expression of a proteininvolved in the transformation of cells and in the presence of acompound being tested for carcinogenicity. Effective conditions include,but are not limited to, appropriate media, temperature, pH and oxygenconditions that permit cell growth. An appropriate, or effective, mediumrefers to any medium in which a cell of the present invention, whencultured, is capable of cell growth and, in the case of a recombinanttest cell, is capable of expression of nucleic acid molecules. Such amedium is typically a solid or liquid medium comprising growth factorsand assimilable carbon, nitrogen and phosphate sources, as well asappropriate salts, minerals, metals and other nutrients, such asvitamins. Culturing is typically conducted in petri plates. Culturing iscarried out at a temperature, pH and oxygen content appropriate for thetest cell. Such culturing conditions are within the expertise of one ofordinary skill in the art.

The assay of the present invention includes contacting a test cell asdescribed above with a compound being tested for carcinogenicity. Forexample, test cells can be grown in liquid culture medium or grown onsolid medium in which the liquid medium or the solid medium contains thecompound to be tested. In addition, as described above, the liquid orsolid medium contains components necessary for cell growth, such asassimilable carbon, nitrogen and micro-nutrients.

The assay disclosed in the present invention involves contacting cellswith the compound being tested for a sufficient time to allow fortransformation of cells in the presence of carcinogenic compounds. Theperiod of contact with the compound being tested can be either theentire growth phase of the assay prior to scoring or some smallerportion thereof. For example, it may be that for more toxic substances ashorter time of contact with the substance being tested is suitable. Asused herein, the term "contact period" refers to the time period duringwhich cells are in contact with the compound being tested. The term"incubation period" refers to the entire time during which cells areallowed to grow prior to scoring. Thus, the incubation period includesall of the contact period and may include a further time period duringwhich the compound being tested is not present but during which growthis continuing prior to scoring.

The incubation time for growth of cells can vary but is sufficient toallow for the development of transformation characteristics intransformed cells. It will be recognized that shorter incubation timesare preferable because compounds can be more rapidly screened forcarcinogenicity. In this regard, test cells used in an assay of thepresent invention, under appropriate growth conditions, will developtransformation characteristics in the presence of carcinogens inpreferably less than about 8 weeks, more preferably less than about 21days, and even more preferably less than about 14 days.

After the incubation period, cell growth is scored for the presence orabsence of one or more transformation characteristic. The appearance oftransformed cells in the present invention, as indicated by the presenceof one or more transformation characteristics, is considered to beindicative that the compound tested by the assay of the presentinvention is likely to be carcinogenic.

In the instance of the transformation characteristic being the formationof foci, cells can be stained and examined visually or with the aid of amicroscope. The presence of foci on culture media indicates the presenceof transformed cells. In a preferred embodiment of using foci formationas the transformation characteristic, test cells are grown with normalcells. As used herein, normal cells are "wild-type" cells, or cells thatdo not have a defect in a protective cellular mechanism as describedherein. Therefore, normal cells do not have the identifyingtransformation characteristics as described above. In this manner, thenormal cells will form a "lawn" or monolayer of contact inhibited cells.If the test compound is a carcinogen, each test cell will lose contactinhibition and grow to form a focus. If the test compound isnon-carcinogenic, the test cells will be contact inhibited just as thenormal cells on the lawn and only a monolayer of cells will be seen.This embodiment of the present invention provides several advantages.The normal cells function as "feeder" cells which condition the mediumand metabolize the compound being tested. Further, the lawn of normalcells provides a background for comparison of transformed foci. Yetanother advantage of the method is that all multi-layered aggregates ofcells which overlay the lawn are counted as foci. In one embodiment, theratio of normal cells to test cells can be between about 100:1 to about1:1, more preferably from about 50:1 to 5:1, and most preferably about10:1.

One advantage of the method of the present invention is that when testcells are exposed to a carcinogen, the cells develop transformationcharacteristics, such as formation of foci, at a rate which isconsidered to be statistically significantly higher than the rate atwhich transformation characteristics are developed in the absence of acarcinogen. More preferably, such cells develop transformationcharacteristics in the presence of carcinogens at a rate about 1 foldgreater (i.e., 100% increase), more preferably about 25 fold greater,and most preferably about 50 fold greater than in the absence ofcarcinogens.

In a further embodiment of the present invention, the occurrence oftransformation characteristics is proportional to the carcinogenicity ofthe compound being tested. That is, the assay method quantifies thecarcinogenicity of the compound being tested. In this manner, therelative carcinogenic potential of two different test compounds at agiven concentration can be evaluated based on the relative occurrence oftransformation characteristics.

One embodiment of the present invention is a method to identifytissue-specific carcinogens. Such method includes contacting a putativetissue-specific carcinogen with a first test cell of a firsttissue-type, and also with a second test cell of a second tissue-type.In this embodiment, both the first cell and the second cell have adefect in a protective cellular mechanism selected from the group of adefect in a DNA damage repair mechanism, a defect in cell cycle controland/or a defect in the ability to prevent damage induced by oxygen freeradicals. An important feature of this embodiment is that the first celland the second cell are of a different tissue-type, but have the samedefect in a protective cellular mechanism as described herein. The term,"tissue-type", as used herein, is described in detail below. The methoddisclosed in this embodiment further comprises scoring cell growth ofthe first test cell and the second test cell based on a transformationcharacteristic as previously described herein. A positive transformationcharacteristic in either of the first or second test cells indicatesthat the compound being tested for carcinogenicity is carcinogenic.Moreover, a difference in the magnitude of a positive transformationcharacteristic between the first and second test cell indicates that thecompound is a tissue-specific carcinogen. Identification oftissue-specific carcinogens is advantageous because the specificcircumstances under which a particular carcinogen is likely to inducecellular transformation can be determined, and because the knowledge ofsuch tissue-specificity can be particularly helpful in research and indeveloping preventative, diagnostic, and/or therapeutic protocols fortreatment of particular diseases.

As used herein, a "tissue-specific carcinogen" refers to a carcinogenwhich preferentially transforms cells of a particular tissue-type overcells of another tissue-type. According to the present invention, a"tissue-type" can refer to a cell-type or, alternatively, to a tissuefrom a particular organ. In one embodiment, a test cell of the presentinvention can be of a cell-type selected from the group which includes,but is not limited to, fibroblasts, epithelial cells, neurons,hepatocytes, keratinocytes, hematopoietic cells, kidney cells, bonecells, and muscle cells. In another embodiment, a test cell of thepresent invention can be derived from a tissue from a particular organ,including, but not limited to, liver, bladder, uterus, bone marrow,lymph node, kidney, pancreas, skin, lung, ovary, colon, stomach,prostate, brain, thyroid, cervix, and parathyroid.

As mentioned above, in this embodiment of the present invention, animportant feature is that the first test cell and the second test cellbe of different tissue types, but have the same defect. For example, thefirst test cell could be a fibroblast having a defect in nucleotideexcision repair, and the second test cell could be a lymphocyte having adefect in nucleotide excision repair (i.e., these cells are of differentcell-types). In another example, the first test cell could be a skinfibroblast having a defect in nucleotide excision repair, and the secondtest cell could be a kidney fibroblast having a defect in nucleotideexcision repair (i.e these cells are from tissues of different organs).In yet another example of this embodiment, a first test cell could be akidney cell of any cell-type which expresses a kidney-specific protein,and the second test cell could be a lung cell of any cell-type whichexpresses a lung-specific protein, wherein both the first and the secondtest cells have a defect in nucleotide excision repair.

The method of identifying tissue-specific carcinogens can use materialsas described generally herein for other methods of the presentinvention. As described above, a specific feature of this embodiment isthat the carcinogen is evaluated for tissue-specificity by determiningif there is a difference in the magnitude of a positive transformationcharacteristic between the first and second test cell, which indicatesthat the compound is a tissue-specific carcinogen. A difference inmagnitude can be any measurable, statistically significant difference ina transformation characteristic between the first and second cell. Sucha difference is determined based on the transformation characteristicbeing used in a particular assay. For example, if the transformationcharacteristic being scored is the formation of foci, and if, afterbeing contacted with a putative tissue-specific carcinogen, the firsttest cell forms significantly more foci than the second test cell, thanthe carcinogen has tissue-specificity for the tissue-type of the firstcell.

In preferred embodiments of the method to identify tissue-specificcarcinogens, more than two test cells can be used in a given assay. Forexample, in a preferred embodiment, a putative carcinogen is contactedwith a third test cell, wherein the third test cell is of a third, anddifferent, tissue-type than either the first or the second test cell,but has the same defect in a protective cellular mechanism as the firstand the second test cell. It is within the scope of this particularembodiment that even more cells of different tissue-types can be used inan assay (i.e., a fourth test cell of a fourth tissue-type, a fifth testcell of a fifth tissue-type, etc.).

Another embodiment of the present invention is a method to identify thebiochemical mechanism of carcinogenicity of a carcinogenic compound. Asused herein, the biochemical mechanism, or biochemical pathway, ofcarcinogenicity of a carcinogenic compound refers to the biochemicalmechanism by which such a compound causes a cell to become transformed.For instance, a particular carcinogen may damage DNA in a cell byalkylating the DNA. If the cell is unable to remove or repair thealkylated DNA, it can become transformed.

A method to identify such biochemical mechanisms of carcinogenicityincludes contacting a putative carcinogen with a first test cell havinga defect in a first protective cellular mechanism, and also with asecond test cell having a defect in a second protective cellularmechanism. In this embodiment, both the first cell and the second cellhave a defect in a protective cellular mechanism which can include adefect in a DNA damage repair mechanism, a defect in cell cycle controland/or a defect in the ability to prevent damage induced by oxygen freeradicals. Such defects include any of the more specific defects in aprotective cellular mechanism as described previously herein. Animportant feature of this embodiment is that the first test cell has adefect in a different protective cellular mechanism than the defect ofthe second test cell.

This method further includes scoring cell growth of the first test celland the second test cell based on a transformation characteristic aspreviously described herein. A positive transformation characteristic ineither of the first or second test cells indicates that the compoundbeing tested for carcinogenicity is carcinogenic. Moreover, a differencein the magnitude of a positive transformation characteristic between thefirst and second test cell indicates that the biochemical mechanism ofthe carcinogen is associated with a specific defect in a protectivecellular mechanism. In other words, if the first test cell becomestransformed in the absence of (or significantly lesser presence of) atransformation characteristic in the second test cell, this result wouldindicate that the biochemical mechanism of carcinogenicity of thecarcinogen being tested is associated with the defect in the protectivecellular mechanism of the first test cell and not with the defect of thesecond test cell.

An example of such a method to identify the biochemical mechanism ofcarcinogenicity of a compound is described as follows. A first test cellcould be a test cell from a patient with Fanconi's anemia which has adefect in the ability to repair DNA damage due to crosslinking, and asecond test cell could be a cell from a patient with xerodermapigmentosum (XP) which has a defect in the ability to repair DNA damagedue to alkylation. A transformation characteristic of a greatermagnitude observed with the first test cell (Fanconi's anemia cell) thanis observed with the second test cell (XP cell), indicates that thebiochemical mechanism of carcinogenicity is by crosslinking of DNA. Onthe other hand, a transformation characteristic of a greater magnitudeobserved with the second test cell (XP cell) than is observed with thefirst test cell (Fanconi's anemia cell), indicates that the biochemicalmechanism of carcinogenicity is by alkylation of DNA.

The method of identifying the biochemical mechanism of carcinogenicityof a compound can use materials as described generally herein for othermethods of the present invention. As previously described herein, adifference in the magnitude of a transformation characteristic between afirst and second cell can be any measurable, statistically significantdifference in a transformation characteristic between the first andsecond cell. Moreover, it is within the scope of this embodiment thatmore than two test cells can be used in a given assay. For example, in apreferred embodiment, a putative carcinogen is contacted with a thirdtest cell having a defect in a third, and different, protective cellularmechanism than either the first or the second test cell. It is withinthe scope of this particular embodiment that even more cells having adefect in different protective cellular mechanisms can be used in anassay.

A further aspect of the present invention is a method to identifyanti-carcinogenic agents. This method can use materials as describedgenerally herein for other methods of the present invention. The methodto identify anti-carcinogenic agents (i.e., transformation inhibitors)can involve the use of test cells which comprise a defect in aprotective cellular mechanism selected from a defect in a DNA damagerepair mechanism, a defect in cell cycle control, and/or a defect in theability to prevent damage induced by oxygen free radicals, as describedin detail above. In one embodiment, this method includes contacting sucha test cell with a known carcinogen. Such a carcinogen can be either agenotoxic or nongenotoxic carcinogen. This method further includescontacting such a test cell in the presence of a carcinogen with acompound to be evaluated for its effectiveness as an anti-carcinogenicagent. Such a cell is contacted with both a carcinogen and a compound tobe tested in the manner as noted above for other methods of the presentinvention. After a suitable incubation period, cell growth is scored forthe presence or absence of one or more transformation indicators asnoted above.

In another embodiment of the method to identify anticarcinogeniccompounds, a test cell as described above, which has the phenotype ofbeing transformed in the absence of a known carcinogen is used. Suchcells have one or more of the transformation characteristics discussedabove.

The absence of a transformation characteristic or a reduction in theincidence of transformation characteristics compared to the rate ofoccurrence of transformation characteristics in the absence of thecompound being tested, is an indication that the compound being testedis effective as an anti-carcinogenic agent.

Carcinogens which can be used in this embodiment of the presentinvention can be any known carcinogen, such as aflatoxin B,3-methylcholanthrene, benzo a!pyrene and 4-aminobiphenyl. Alternatively,carcinogens can be any other known carcinogen or carcinogens identifiedin the future.

The foregoing discussion of the invention has been presented forpurposes of illustration and description. Further, the description isnot intended to limit the invention to the form disclosed herein.Consequently, variation and modification commensurate with the aboveteachings, within the skill and knowledge of the relevant art, arewithin the scope of the present invention. The embodiment describedhereinabove is further intended to explain the best mode presently knownof practicing the invention and to enable others skilled in the art toutilize the invention as such, or other embodiments, and with thevarious modifications required by their particular application or usesof the invention. It is intended that the appended claims be construedto include alternative embodiments to the extent permitted by the priorart.

What is claimed is:
 1. A method to identify tissue-specific carcinogens,said method comprising:(a) contacting a putative tissue-specificcarcinogen with a first test cell of a first tissue-type, wherein saidfirst test cell has a defect in a protective cellular mechanism; (b)contacting said putative tissue-specific carcinogen with a second testcell of a second tissue-type, wherein said second test cell has the samedefect as said first test cell; and (c) scoring cell growth of saidfirst test cell and said second test cell based on a phenotypictransformation characteristic, wherein a positive transformationcharacteristic in either of said first or second test cells indicatesthat said putative tissue-specific carcinogen is carcinogenic, andwherein a difference in the magnitude of a positive transformationcharacteristic between said first and second test cells indicates thatsaid putative tissue-specific carcinogen is a tissue-specificcarcinogen.
 2. The method of claim 1, wherein said defect in aprotective cellular mechanism is selected from the group consisting of adefect in a DNA damage repair mechanism, a defect in cell cycle control,and a defect in the ability to prevent damage induced by oxygen freeradicals.
 3. The method of claim 1, wherein said first and secondtissue-type are a cell-type selected from the group consisting offibroblasts, epithelial cells, neurons, hepatocytes, keratinocytes,hematopoietic cells, kidney cells, bone cells, and muscle cells, andwherein said first tissue-type and said second tissue-type aredifferent.
 4. The method of claim 1, wherein said first and secondtissue-type are a tissue from an organ selected from the groupconsisting of liver, bladder, uterus, bone marrow, lymph node, kidney,pancreas, skin, lung, ovary, colon, stomach, prostate, brain, thyroid,cervix, and parathyroid, and wherein said first tissue-type and saidsecond tissue-type are different.
 5. The method of claim 1, furthercomprising contacting said putative tissue-specific carcinogen with athird test cell of a third tissue-type having the same defect as saidfirst and second test cells, wherein said third tissue-type is differentthan said first tissue-type and said second tissue-type, and scoringcell growth of said third test cell based on a transformationcharacteristic.
 6. The method of claim 1, wherein said defect is adefect in a DNA damage repair mechanism selected from the groupconsisting of an inability to perform direct DNA repair, an inability toperform base excision repair, an inability to perform nucleotideexcision repair, an inability to perform nucleotide mismatch repair, aninability to perform post-replication repair, an inability to performcross-link repair, an inability to perform double-strand break repair,and an inability to perform transcription repair.
 7. The method of claim1, wherein said test cell has a defect in cell cycle control, andwherein said defect is a defect in a component involved in a cellularmechanism controlling cell cycle.
 8. The method of claim 1, wherein saiddefect is a defect in the ability to prevent damage induced by oxygenfree radicals, and wherein said defect is a defect in a componentinvolved in control of an oxidative stress response.
 9. The method ofclaim 1, wherein said test cell is a human cell, and wherein said humancell is derived from a patient having a disease selected from the groupconsisting of xeroderma pigmentosum, Cockayne's syndrome,trichothiodystrophy, Fanconi's anemia, ataxia-telangiectasia, hereditarynonpolyposis colon cancer, promyelocytic leukemia, lymphoid leukemia,myeloid leukemia, colorectal carcinoma, amyotrophic lateral sclerosis,Li-Fraumeni syndrome, and Bloom's Syndrome.
 10. The method of claim 1,wherein said test cell is a human cell, and wherein said human cell isengineered to have said defect.
 11. The method of claim 1, wherein saidtransformation characteristic is selected from the group consisting offormation of foci, loss of growth factor or serum requirements, andanchorage independence.